Corneodesmosin: Structure, Function and Involvement in Pathophysiology Nathalie Jonca, Cécile Caubet, Marina Guerrin, Michel Simon and Guy Serre*

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36 The Open Dermatology Journal, 2010, 4, 36-45 Open Access Corneodesmosin: Structure, Function and Involvement in Pathophysiology Nathalie Jonca, Cécile Caubet, Marina Guerrin, Michel Simon and Guy Serre*

CNRS – University Toulouse III, UMR 5165 “Epidermis Differentiation and Rheumatoid Autoimmunity”, IFR 150 (INSERM-CNRS-University Toulouse III-CHU), France

Abstract: Corneodesmosin (CDSN) was identified in the early 90th by raising monoclonal antibodies against human plantar stratum corneum. It is a protein specific to desmosomes that will undergo transformation into corneodesmosomes, i.e. in man, desmosomes of the epidermis, of the three epithelial layers of the inner root sheath of the hair follicles and of the hard palate epithelium. After its secretion by granular keratinocytes via the lamellar bodies, CDSN is incorporated into the desmoglea of the desmosomes shortly before their transformation into corneodesmosomes. CDSN displays adhesive properties, mostly attributable to its N-terminal glycine-rich domain, and is sequentially proteolyzed as corneocytes migrate towards the skin surface. The recent inactivation of Cdsn in mice induced a lethal epidermal barrier disruption and hair follicle degeneration related to desmosome dysfunction, confirming the essential role of the protein in epidermis and hair follicle integrity. CDSN is located on chromosome 6, in the major psoriasis susceptibility locus PSORS1. Intriguingly, the only monogenic disease identified so far associated with nonsense mutations in CDSN, leading to the formation of a truncated protein, is a rare autosomal dominant disease, hypotrichosis simplex of the scalp. In this review, we expose data from the discovery of the protein to the most recent findings related to the relationship between its structure and function. In particular, the important benefits of mouse models and human diseases for the comprehension of CDSN role in the epidermis and hair follicles are reported in details. Keywords: Corneodesmosome, corneodesmosin, cornification, desquamation, differentiation, epidermis, hyperkeratosis, hypotrichosis, kallikrein, psoriasis, stratum corneum.

INTRODUCTION desmosomes in the SC, contributed to reconsider the place of proteins in the barrier function of the SC. The aim of epidermal differentiation is to produce an efficient barrier separating the body from the environment. This review focuses on CDSN. We expose the data from Such a role is fulfilled by the most superficial part of the the discovery of the protein to the more recent findings epidermis, the stratum corneum (SC). This layer is related to the relation between the structure and function of composed of corneocytes, which are dead and flattened CDSN. In particular, the important benefits of mouse models “mummified” cells endowed with a cornified envelope that and human diseases for the comprehension of the primordial provides extreme individual resistance. Owing to its relative role of CDSN in the epidermis and hair follicles are reported impermeability to water and water soluble substances, the in details. SC provides an efficient “inside-out” and “outside-in” CDSN: DISCOVERY OF A NEW PROTEIN SPECIFIC barrier. Its physical resistance and strong cohesion, TO THE LATE STEPS OF EPIDERMAL DIFFER- combined with its constant renewal, also offers a barrier ENTIATION against physical, chemical and microbial agents from the environment. In the 80th, the predominant model to explain In the early 90’s, Guy Serre’s group raised monoclonal the barrier function of the SC was the “brick wall” model antibodies against plantar SC in order to identify new late proposed by P. Elias, in which the corneocytes were the markers of epidermal differentiation. This led to the bricks endowed within an intercellular substance composed identification of CDSN, specifically recognized by two mostly of lipids. In the early 90th, emerging data suggested monoclonal antibodies, G36-19 and F28-27. Immuno- that the lipids, crucial for the barrier’s impermeability, histological studies indicated that CDSN is mainly expressed played only a limited part in tissue cohesion, whereas the in cornified epithelia, and is absent from the non-cornified integrity of the SC depended mostly on the corneocytes and epithelia of the vagina, uterine cervix, or esophagus [3]. In associated intercellular junctions. In particular, the finding addition, CDSN is present in the thymus Hassal’s bodies, that protease activity was necessary for desquamation to take containing epithelial cells very similar to differentiated place [1, 2], as well as the identification of corneodesmosin epidermal keratinocytes, and the CDSN gene was shown to (CDSN) as the only protein specifically localized in the be transcribed in the placenta by RT-PCR experiments [4]. extracellular part of corneodesmosomes [3], the modified CDSN is also expressed in the hair follicle. The protein appears asynchronously in the 3 compartments of the inner

root sheath, as their cells cornify: first in the Henle’s layer, *Address correspondence to this author at the CNRS-UPS UMR5165, CHU then in the cuticle, and lastly in the Huxley’s layer [3, 5]. Purpan, Place du Docteur Baylac, TSA 40031, 31059 Toulouse Cedex 9, CDSN is also clearly detected in cells of the hair follicle France; Tel: 33 5 6115 8402; Fax: 33 5 6149 9036; E-mail: [email protected] medulla [4].

1874-3722/10 2010 Bentham Open Corneodesmosin in Normal and Diseased Skin The Open Dermatology Journal, 2010, Volume 4 37

In the epidermis, as shown by immunohistology, CDSN covalent links. The nature of these covalent links, the is cytoplasmic in the lower stratum granulosum (SG) then it molecular partner -protein or lipid- involved in these links, becomes pericellular and progressively disappears in the as well as the enzyme responsible for the crosslink, remain lower SC. In contrast, the protein persists up to the unknown. Transglutaminases 1, 3 and 5 are responsible for desquamating keratinocytes in the palmoplantar epidermis the crosslink of the envelope precursor in the granular and hard palate epithelium, with a discontinuous keratinocyte. However, these enzymes are located in the pericorneocyte microgranular distribution. Similarly, CDSN cytosol or anchored to the internal side of the cytoplasmic is detected as surface spots on corneocytes isolated by membrane. No transglutaminase isoform was described to be scraping the hard palate or the epidermis. Immunoelectron secreted in the extracellular space, where it could use CDSN microscopy reveals that CDSN is present in the lamellar as a substrate. CDSN is the first and until now the only bodies from their emergence in the upper SG, in the protein specifically localized in the extracellular core of extracellular space of the desmosomes of the granular corneodesmosomes. This discovery contributed to the idea keratinocytes and lastly in the core of the modified that corneodesmosomes are not only desmosome remnants, desmosomes of the SC. Finally, CDSN was shown to be as considered until then, but fulfill a precise function in the largely preserved at the surface of purified cross-linked upper layers of the epidermis. envelopes, where it is located on fibrils gathered over the CDSN is a 52-56 kDa basic phosphoprotein. external side (Fig. 1). Moreover, as the envelopes were Deglycosylation experiments, reactivity to lectins, and purified under reducing and denaturing conditions, these data chromatography on concanavalin A-sepharose indicated that demonstrate that CDSN is covalently linked to the cornified it is N-glycosylated, with the oligosaccharide moiety envelope not only by disulfide bonds but also by other comprising ~10% of the protein mass [6]. Cloning of its

Fig. (1). Localization of CDSN in human epidermis and hair follicle. (A) Immunolocalization of CDSN in human breast epidermis by indirect immunofluorescence. (B-D). Immunoelectron microscopy analysis reveals CDSN in lamellar bodies and in the intercellular part of desmosomes (arrow) of the granular keratinocytes (B) as well as in the core of corneodesmosomes in the SC (C). CDSN is also bound to the cornified cell envelopes (D). (E, F). Immunofluorescence staining on transversal (E) and longitudinal (F) sections of hair follicles shows a restricted labeling of CDSN in the three compartments of the inner root sheath: Henle’s layer (He), Huxley’s layer (Hu) and cuticle (cut), and in the medulla (med). Scale bars: 50 m (A), 200 nm (B, C), 100 nm (D), 20 m (E, F). In (A), nuclei were counterstained with Toto-3; dotted line, dermoepidermal junction. From references [3, 4] and unpublished data. Scale bars: 50 m (A), 200 nm (B, C), 100 nm (D), 20 m (E, F). In (A), nuclei were counterstained with Toto-3; dotted line, dermoepidermal junction. From references [3, 4] and unpublished data. 38 The Open Dermatology Journal, 2010, Volume 4 Jonca et al. encoding cDNA revealed that CDSN is located on CDSN displays adhesive properties mediated by its N- chromosome 6, in PSORS1, the major locus for psoriasis terminal glycine loop domain. susceptibility. The mRNA encodes a 529-amino acid protein with a N-terminal signal peptide and one putative N- THE PROGRESSIVE PROTEOLYSIS OF CDSN IN THE STRATUM CORNEUM IS A PRE-REQUISITE glycosylation site, consistent with the demonstration that it is secreted and glycosylated [7]. FOR DESQUAMATION As a step in elucidating the mechanisms of tissue-specific A tight balance between keratinocyte proliferation in the expression, the human CDSN promoter was characterized basal layer and cell shedding at the surface of the epidermis using transgenic mice. The transgene consisted on the 4.2 kb is essential for skin homeostasis and renewing of the SC upstream region of the human CDSN transcription initiation protective barrier. Ultrastructural studies demonstrated that site linked to the LacZ gene. The reporter gene expression the degradation of corneodesmosomes in the outermost was detected by histoenzymology in special areas of the corneocytes is concomitant to desquamation, and showed inner root sheath of the hair follicles and the hair medulla, that proteolytic cleavage of the extracellular part of these with an expression pattern that perfectly matched that of the cell-cell adhesive structures is a key event in this process. In endogenous protein. The 4.2 kb DNA fragment is thus non-palmoplantar SC, normal exfoliation of corneocytes sufficient to correctly drive the expression of the reporter results from 2 steps of corneodesmosomal degradation. First, gene in the hair follicle. Surprisingly, no -galactosidase the non-peripheral corneodesmosomes are degraded at the activity was detected in the granular keratinocytes of the interface between the stratum compactum and the stratum epidermis. However, induction of epidermal disjunctum. Second, the persistent peripheral corneodesmo- hyperproliferation either by pharmacological agents in topic somes are broken at the skin surface allowing corneocyte or by wounding, led to strong expression of the reporter gene separation. Desquamation in palmoplantar skin is quite in the keratinocytes of the SG and in the parakeratotic different, as the non-peripheral corneodesmosomes appear to corneocytes of the SC. This suggests that the transgene did be preserved against proteolysis in the lower SC accounting not comprise all the elements necessary for the expression of for the formation of a thick cornified layer, characteristic of CDSN in normal epidermis [4]. these anatomic regions. This persistence is also a common feature of ichthyosis [15, 16]. CDSN IS AN ADHESIVE PROTEIN The presence of cleavage products of CDSN in extracts A striking feature of CDSN is its very high serine and of superficial SC attests to the degradation of glycine content (27.5% and 16%, respectively), particularly corneodesmosomes. When extracted from viable layers of at both termini of the protein, i.e. amino acids 60-171 and human epidermis, CDSN showed an apparent molecular 375-450. Similar serine- and glycine-rich domains described mass of around 52-56 kDa, whereas a molecular form of 33 at both ends of keratins have been proposed to form kDa is the major form extracted from the most superficial structural motifs called “glycine loops” as a consequence of and less firmly attached corneocytes [3]. The presence of the association of interspersed aromatic or aliphatic residues. CDSN in the extracellular core of corneodesmosomes, and These glycine loop domains have been suggested to mediate the in vitro demonstration of the homophilic adhesive intermolecular adhesion by acting like a Velcro [8]. Such properties of the protein, strongly suggest that, in vivo, molecular interactions could be essential for skin CDSN contributes to the strong cohesion between homeostasis since mutations in the glycine loops of keratins corneocytes. Consequently, its proteolysis was proposed as and loricrin lead to various human cutaneous diseases, e.g., one of the major biochemical changes that lead to ichtyosis hystrix and palmoplantar keratodermas [9-12]. desquamation [17]. The potential role of CDSN in homophilic cell-cell Deglycosylation experiments and reactivity with lectins interactions was assessed with mouse L-fibroblasts demonstrated that CDSN carbohydrate moiety does not expressing a chimeric protein composed of the human CDSN prevent the protein proteolysis. Using a set of affinity- and the transmembrane and cytoplasmic domains of mouse purified anti-peptide antibodies and monoclonal antibodies E-cadherin. CDSN thus anchored at the cell surface mediates recognizing the five structural domains of CDSN, a refined cellular aggregation. Overlay binding assays and quantitative characterization of its proteolysis during terminal analysis by surface plasmon resonance using bacterial differentiation of epidermis was realized. Immunoblotting, recombinant forms of full-length CDSN confirmed the immunohistochimistry and immunoelectron microscopy homophilic adhesive properties of the protein [13, 14]. experiments revealed that CDSN processing begins in the Moreover, recombinant CDSN associates into large homo- SG. In an initial proteolysis step, the N- and C-terminus of oligomers of at least three subunits that are only partially the protein are eliminated, giving rise to a 48-46 kDa CDSN dissociated in 8 M urea. These very stable oligomers fragment that incorporates into desmosomes and might obtained in vitro possibly correspond to cis- and/or trans- contribute to their transformation in corneodesmosomes. interactions in vivo. Similar experiments of size-exclusion This fragment is still endowed with the glycine loop-related chromatography and surface plasmon resonance performed domains. Later, at the transition between the stratum with truncated forms of recombinant CDSN showed that the compactum and the stratum disjunctum, cleavage of the N- N-terminal glycine loop domain, that perfectly matches terminal adhesive glycine loop domain of the protein leads to features of the Steinert’s glycine loop model, was necessary a 36-30 kDa fragment. This step seems to be correlated with and sufficient, and thus responsible for both the the abrupt decrease in SC cohesion. Finally, a CDSN oligomerization of CDSN and its homophilic adhesive fragment of only 15 kDa, mainly corresponding to the properties. Altogether, these data clearly establish that central part of the protein and devoid of both N- and C- Corneodesmosin in Normal and Diseased Skin The Open Dermatology Journal, 2010, Volume 4 39

Fig. (2). Schematic representation of CDSN proteolysis during the terminal differentiation of epidermis. A schema of CDSN proteolysis from the 52-56 kDa protein to a fragment of 15 kDa is proposed as deduced from immuno-ultrastructural and biochemical data. The apparent molecular mass of the various CDSN forms are indicated, the position of the epitopes recognized by the anti-CDSN anti- peptide sera (A40–55, B102–115, C409–423, and D472–486) and monoclonal antibodies (G36-19 and F28-27) are shown by black boxes. The glycine loop-related domains and the N-glycosylation site of CDSN are represented by wavy lines and a black dot, respectively. From reference [18]. terminal glycine-rich domains, is present at the surface of the namely desmocollin 1 (DSC1) and desmoglein 1 (DSG1), by non-cohesive corneocytes [6, 18] (Fig. 2). KLK5 and KLK7 was realized. KLK7 directly cleaved A number of different proteases of the serine, cysteine, or CDSN and DSC1 but was unable to degrade DSG1. Incubation with KLK5 induced degradation of the three aspartic protease families, as well as protease inhibitors of corneodesmosomal components. Moreover, it was shown these classes of enzymes, have been identified in the SC and that KLK7 and KLK5 conserved their activity at pH 5.6, might play a role in desquamation (for a review see [19-21]). close to that of physiological conditions in the SC. This Among these proteases, KLK7 and KLK5 - also known as study also suggested that KLK5 was able to activate the stratum corneum chymotryptic enzyme (SCCE) and stratum corneum tryptic enzyme (SCTE), respectively- are serine proform of KLK7 [27]. proteases of the kallikrein family [22]. They are both highly It is highly probable that, besides serine proteases, the expressed in granular keratinocytes and present in the involvement of other classes of proteases, also present in the intercellular spaces of the SC. Synthesized as inactive SC, contribute to corneodesmosomal components proforms, they are both activated by cleavage of a short N- degradation. Among these are the 2 cysteine proteases, terminal domain performed by an enzyme with the Cathepsin L2 (CTSL2, also named Cathepsin V) and characteristics of a trypsin-like protease [23, 24]. They were Cathepsin L-like (CTSL-like), and one aspartic acid protease proposed to be actors of a cascade of activated proteases and Cathepsin D (CTSD). Synthesized as pro-enzymes, inactive pro-enzymes that could regulate the rate of cathepsins undergo proteolytic maturation, sometimes in an desquamation [25] (for a review see [21]). The importance of autocatalytic way, and are active in an acidic environment. pH for SC cohesion and desquamation was also evidenced They have long been classified as hydrolases with exclusive using superbases topically applied on hairless mouse skin functions in terminal degradation of proteins in the [26]. CDSN was demonstrated to be a preferred substrate of lysosomal compartment. Active forms of CTSL-like and both serine proteases in vitro [18]. A detailed analysis of the CTSD have been shown to cleave CDSN in vitro ([28, 29] proteolysis of CDSN, but also of two other major and unpublished results). A detailed description of proteases components of the extracellular core of corneodesmosomes, and protease inhibitors, as well as recent data on the 40 The Open Dermatology Journal, 2010, Volume 4 Jonca et al. degradation of the corneodesmosomal components and on transition. At the ultrastructural level, the main part of desquamation, leading to a model of regulation of numerous split junctions typically remain attached to the corneodesmosomal components degradation, has been granular keratinocytes, suggesting that the cohesive defect recently reviewed elsewhere [20]. lies in the upper side of the junction (Fig. 4). Thus, although Cdsn was already present in the extracellular core of ESSENTIAL ROLE OF CDSN: LESSONS FROM desmosomes from the SG, it seemed to play its fundamental ANIMAL MODELS role only when cornification is complete. Intriguingly, no The demonstration that CDSN is an adhesive protein thinning out of single or bundled corneocytes throughout the covalently linked to the cornified envelope, as well as the SC was observed but detachment of the whole SC from the data showing that its sequential proteolysis seems to be a subjacent SG. This suggested that the SG/SC interface may prerequisite to desquamation, strongly suggest that CDSN is actually be the most fragile zone as it links two epidermal primordial for corneodesmosome function. But how exactly layers with different junctional organizations and mechanical the protein fulfils its function remains unclear. CDSN was characteristics: desmosomes and keratin intermediate proposed to be necessary for corneodesmosome filaments organized in taut cables in the SG, rigid cornified morphogenesis. It could also protect the other envelopes linked by corneodesmosomes in the SC. corneodesmosomal components from premature degradation by the proteases involved in desquamation. Finally, CDSN could reinforce cohesion by its own adhesive properties. Analysis of mouse models with an inactivated Cdsn gene is of particular interest to answer these questions. The first publication of Cdsn inactivation in mice, obtained by insertional mutation, concluded that Cdsn is necessary for corneodesmosome formation [30]. We also realized conditional ablation of Cdsn using the K14-driven Cre- mediated recombination. The detailed analysis of our Cdsn deficient mice confirmed that Cdsn is vital for epidermal barrier and hair follicle integrity, and our conclusion supported the idea that Cdsn is mainly an adhesive protein [31]. Immediately after birth, KO neonates showed a severe skin detachment starting from ventral area and extremities (paws and snoot), and died within 1h. In contrast, heterozygous newborns did not develop any skin phenotype, demonstrating that ablation of only one allele had no consequences. Surprisingly, KO pups obtained after caesarian delivery at E18.5 were indistinguishable from their littermates. However, during grooming by surrogate mother, they rapidly developed a skin phenotype similar to that observed after natural birth (Fig. 3). The effects of the mutation on skin permeability were analyzed by dye penetration assay and transepidermal water loss measurements, and the mechanical resistance of the skin was evaluated by quantification of SC proteins obtained by tape stripping. The results showed that, although the greatly reduced mechanical resistance of the SC is an intrinsic feature caused by Cdsn deficiency, epidermal tear, leading to lethal barrier defect, occurred only under mechanical stress encountered after birth. Fig. (3). Mice lacking Cdsn show a drastic skin tear leading to Intact dorsal skin of Cdsn KO newborns (i.e. before postnatal death. (A) Macroscopic appearance of wild type (WT), appearance of the macroscopic phenotype) observed by heterozygous (HET) and knockout (KO) pups born at full term. KO transmission electron microscopy revealed a similar pups died within 1 h after birth and showed considerable skin organization and thickness of the granular layer and the SC detachment, particularly in the ventral area, paws and snout while than WT skin. In particular, we did not detect any significant HET newborns had no phenotype. (B) Macroscopic appearance of differences in the number of transitional desmosomes WT, HET and KO mice delivered by caesarian section at E18.5. between WT and KO neonates, unlike Matsumoto and Immediately after birth, KO neonates were indistinguishable from coworkers’ published data [30]. Moreover, the electron the WT and HET littermates. (C) When neonates were left in density of the corneodesmosomes appeared unchanged, surrogate mother’s care for 1h, the skin phenotype of KO pups suggesting no premature degradation of these structures. became manifest and death occurred rapidly. Higher magnification Thus, Cdsn does not seem indispensable for the of paw and snout of WT and KO neonates are shown on the right morphogenesis of corneodesmosomes nor their protection panel. From reference [31]. from SC proteases. On the other hand, the histological In order to assess long-term consequences of Cdsn analysis of dorsal KO skin revealed the presence of blisters inactivation, we performed grafting of skin from Cdsn- as soon as cornification occurs, that is, at the SG/SC deficient newborns onto nude mice. The grafted epidermis Corneodesmosin in Normal and Diseased Skin The Open Dermatology Journal, 2010, Volume 4 41

Fig. (4). Ultrastructural abnormalities in CDSN knockout neonate epidermis. Dorsal skin of WT and KO mice was processed for transmission EM. (A, B) At low magnification, the overall morphology of the cells at the junction between the granular and cornified layers shows no obvious abnormality. (G, G1, G2), granular keratinocytes; (C1) to (C9), corneocytes, with C1 being adjacent to the granular layer and C9 being nearest the skin surface. (C-H) The ultrastructure of desmosomes and corneodesmosomes from WT (C-E) and KO (F-H) epidermis was analyzed at higher magnification. Regular desmosomes in the SG (C, F), transitional desmosomes at the SG/SC interface (D, G), and corneodesmosomes (white arrows) in the SC (E, H), show the same classical morphology in WT and KO epidermis. (I, J) Detachment between granular keratinocyte and corneocyte is frequently observed in KO epidermis. Split desmosomes isolated (I) or adjacent (J) can be seen. The mid-dense intercellular plugs of the detached transitional desmosomes remain associated with the granular keratinocyte (black arrowhead) whereas an interruption in the electron dense line corresponding to the cornified envelope is clearly visible at the surface of the corneocyte, at the site where the desmosome was attached (white arrowhead). Bars: A, B = 2 m, C-J = 0.2 m. From reference [31]. 42 The Open Dermatology Journal, 2010, Volume 4 Jonca et al. first developed acanthosis and hyperkeratosis. Increased polymorphic, with more than one SNP every 100 bp in the expression of various differentiation markers (involucrin, coding region [35]. Some of these SNPs have been K10), and induced expression of the hyperproliferative associated with psoriasis in many genetic studies (reviewed keratin K6, confirmed the altered differentiation and the in [36]). However, PSORS1 spans a ~300 kb region hyperproliferative state of the grafted epidermis. However, containing at least 11 other genes including HLA-C, and the these compensatory mechanisms appeared to be ineffective, exact identity of the PSORS1 gene remains controversial and were followed by a complete disappearance of the owing to extensive linkage disequilibrium across this region. epidermis. Cdsn was thus necessary for maintaining the A recent study revealed that only 2 genes from the locus, integrity and barrier function of the postnatal epidermis. The HLA-C and CDSN, yield alleles encoding protein unique to consequences of Cdsn loss in adult mice was investigated risk haplotypes [37]. However, this study and others, using using an additional model consisting on K14-driven Cre- ancestral haplotype analysis, resulted in contradictory mediated loxP recombination system with a chimeric findings for the PSORS1 gene: HLA-C, CDSN, or both genes CreERT2 recombinase that can be induced by tamoxifen. [37-41]. Ultrastructural analyses of psoriatic skin show that Inducible Cdsn KO resulted in histological abnormalities desmosomes are not dramatically transformed into similar to that of the Cdsn KO skin graft model. Barrier corneodesmosomes at the SG / SC transition, but rather stay restoration remained unsuccessful, compromising the vital at an intermediate state between the two structures. As prognosis of the mice when the whole skin area was already stated, persistence of corneodesmosomes affected. Thus, Cdsn seemed to play a vital role in adult accompanied by an increased amount of CDSN in the SC, is epidermis too. a feature common to hyperkeratotic diseases. However, the earlier synthesis and secretion of CDSN in the upper stratum These somatic and inducible Cdsn KO mice also allowed spinosum, is specific to psoriasis among several to investigate the consequences of Cdsn loss on hair follicles. hyperproliferative disorders [34]. Finally, persistence of full- Analysis of skin sections from newborn Cdsn KO mice showed a similar morphology and number of hair follicles to length CDSN and of other corneodesmosomal proteins in the upper SC was observed in psoriatic lesions [42]. The those of skin from WT littermate, suggesting that Cdsn psoriasis-associated SNPs of CDSN could affect the function seemed to be dispensable for hair follicle morphogenesis. of the corresponding proteins. To date, only one study has Long term consequences of Cdsn excision in hair follicle shown that an intragenic SNP (SNP*971T), present in 3 was analyzed after grafing of dorsal Cdsn KO skin onto nude common haplotypes associated with psoriasis in various mice and in adult mouse skin induced for Cdsn excision. In both cases, hair follicles first showed an altered morphology, ethnic groups, is responsible for an increased stability of the encoded mRNAs [43]. Although these results are in developed cysts, and finally disappeared. This suggested that agreement with the known overexpression of CDSN in Cdsn is necessary for maintaining normal hair follicle psoriatic lesions [15, 34], they pointed a discrepancy architecture. between CDSN haplotypes at the mRNA level only. The Altogether, these data obtained in vivo demonstrated that CDSN*TTC allele (SNP*619T, SNP*1236T, SNP*1243C), Cdsn plays a vital role in the epidermal barrier and hair associated with the disease in many different ethnic groups, follicle integrity by preventing the rupture of corneodesmo- was identified as the smallest combination of protein altering some. variations unique to CDSN carried by risk haplotypes [37]. Assessment of the functional consequence of these amino- CDSN AND HUMAN DISEASES acid substitutions should provide insight into CDSN putative A common feature of numerous skin disorders is scaling, involvement in psoriasis (Jonca et al., submitted). usually associated with thickening of the SC. The Intriguingly, the only monogenic disease associated with hyperkeratosis may be due to increased cell proliferation, mutations in CDSN that has been identified so far revealed a impaired desquamation, or a combination of both. hair phenotype and did not affect the epidermis. Indeed, Hyperkeratosis has been invariably associated with the nonsense mutations in CDSN were found to be responsible persistence of both peripheral and non-peripheral for a rare autosomal dominant disease, hypotrichosis simplex corneodesmosomes in the outer SC, a feature reminiscent of of the scalp (HSS; OMIM 146520) [44]. Affected normal palmoplantar epidermis. This has been accurately individuals experience progressive loss of scalp hairs described in the case of several congenital ichthyoses [32, beginning in the middle of the first decade and almost 33], winter xerosis [16] or soap-induced xerosis [19]. In complete baldness by the third decade. The body hairs, parallel, increased amounts of CDSN were detected in all the beard, eyebrows and axillary hairs are normal. To date, 3 analyzed hyperkeratotic lesions, including inflammatory different nonsense mutations have been reported in 4 diseases [15, 34]. Mutations in several different genes were families from Israel, Denmark and Mexico [45]. All of them identified as responsible for ichtyosis, but none of them, to produce a truncated form of CDSN, mainly corresponding to date, corresponded to mutations in CDSN. As a defect in the the N-terminal glycine loop domain. An antibody directed barrier is commonly encountered in hyperkeratosis, CDSN against this domain labeled irregularly sized clusters located over-expression occurring in the epidermis from affected in ridges of the superficial dermis and at the periphery of the individuals may rather result from a compensatory hair follicles deeper in the dermis. These aggregates did not mechanism to the barrier defect. stained with several antibodies directed against epithelial CDSN is of particular interest in relation to psoriasis. proteins (like keratins K1, K2, K1O, K11, (pro)filaggrin or Indeed, as previously mentioned, CDSN is localized to involucrin) strongly implying that in these aggregates the chromosome 6p21 at PSORS1, the major susceptibility locus truncated CDSN is not associated with any of these proteins. of this chronic inflammatory skin disorder. CDSN is highly By western blot analysis of dermis extracts, the truncated Corneodesmosin in Normal and Diseased Skin The Open Dermatology Journal, 2010, Volume 4 43

CDSN was detected as SDS- and boiling-resistant dimers, onset of alopecia and the fact that lost hairs are not trimers and larger oligomers [44]. regenerated, another possibility is that the observed CDSN aggregates are toxic for the hair follicle cells. The aggregates We noticed histological similarities between HSS and the released from defective hair follicles probably migrate two main forms of primary localized cutaneous amyloidosis, namely lichen amyloidosis and macular amyloidosis, two through the dermis until they reach the dermo-epidermal junction under which they accumulate. As similar aggregates rare chronic pruritic skin disorders. Amyloidoses form a are not detected in the epidermis, these are probably large heterogeneous group of misfolding diseases associated degraded by SC proteases. with the conversion of soluble peptides/proteins into highly organized fibrillar aggregates [46]. Shape, tinctorial The results from our observations on affected individual properties and secondary structure of amyloid fibrils are skin biopsies, together with the data from experiments characteristic: the fibrils are unbranched, 7.5-20-nm thick, performed in vitro using recombinant mutant forms of bind Congo Red and Thioflavine T dyes, exhibit yellow- CDSN or the N-terminal glycine/serine rich domain of green birefringence in polarized light upon binding of Congo CDSN alone revisits HSS as a possible amyloidosis, among Red, and have a significant content of -sheets that are the growing list of these human diseases. systematically hydrogen-bonded. In both lichen amyloidosis and macular amyloidosis, amyloid material that could be CONCLUSION – FUTURE PROSPECTS derived from epidermal keratins accumulates in the dermal The data accumulated from the discovery of CDSN to the papillae [46-52]. With regard to the histological similarities more recent works using mouse models or studying human between HSS and these two localized cutaneous diseases led to the comprehension of the primordial role of amyloidoses, we wondered if HSS was indeed an CDSN in epidermis and hair follicle integrity. However, amyloidosis (Caubet et al., submitted). Consistent with the some functional aspects concerning CDSN are not fully possibility that the mutant CDSN may adopt an amyloid understood, especially at the molecular level. For instance, conformation, the dermal deposits from skin biopsies of immunoelectron microscopy experiments clearly affected individuals were stained by Thioflavine T and demonstrate that CDSN exclusively localizes to the Congo Red. The presence of the SAP component, a protein extracellular core of corneodesmosomes in the SC. But how that colocalizes with amyloid plaques, irrespective of their is this specific localization achieved? It is likely that a pre- chemical nature and the clinical type of amyloidosis, was existing component of the extracellular part of the junction also evidenced within CDSN deposits. In agreement with interacts with CDSN. We were unable to evidence any these in vivo observations, analysis by electron microscopy interaction between CDSN and desmosomal cadherins demonstrated that recombinant forms of the truncated (unpublished results). The molecular partner for CDSN thus mutated CDSN expressed in the skin of HSS patients, as remains to be identified. Similarly, the molecular event, well as the recombinant N-terminal glycine/serine rich probably involving transglutaminase-like enzymatic activity, domain of CDSN alone, did assemble into ring-shaped and resulting in the covalent link of CDSN to the cornified oligomeric structures and into fibrils. The amyloid-like envelope, has not yet been characterized. Finally, the structure of the fibrils was evidenced by tinctorial affinity for underlying molecular mechanism by which CDSN assumes Congo Red and Thioflavine T dyes. The recorded Fourier its adhesive function within the corneodesmosome has not transform infrared spectra suggested that the recombinant N- been fully elucidated. The previously demonstrated terminal glycine/serine rich domain of CDSN assembly into homophilic adhesive properties of CDSN and the strong fibrils was accompanied by a conformational change with a resistance of aggregates formed by bacterially recombinant very significant increase in -sheet content typical of the CDSN to highly denaturing conditions [13, 14] suggested assembly of native unfolded polypeptides into amyloid that, in vivo, CDSN reinforced cohesion by its own adhesive fibrils. Finally, the truncated CDSN forms expressed in the properties. However, adhesion provided by glycine-loop skin of HSS patients, and assembled into pre-fibrillar domains has been suggested to mediate reversible and oligomers, were toxic to primary keratinocytes grown in constantly adjustable intermolecular links [8]. Consistent vitro. This was consistent with two recent reports suggesting with this, CDSN could give the junction the elasticity that soluble oligomeric species rather than mature amyloid essential to prevent breaking as soon as the cell envelope fibrils were the toxic entity in amyloid-related diseases [53, rigidifies. Clarification of those molecular aspects of CDSN 54]. is the task for the future. Mouse models with inactivation of Cdsn showed that REFERENCES heterozygous Cdsn+/- mice were indistinguishable from their wild type littermate. In particular, they did not develop any [1] Bissett DL, McBride JF, Patrick LF. Role of protein and calcium in stratum corneum cell cohesion. Arch Dermatol Res 1987; 279: 184- hair phenotype up to 8 months. This reinforced the 9. hypothesis that HSS is not caused by Cdsn [2] Lundstrom A, Egelrud T. Stratum corneum chymotryptic enzyme: haploinsufficiency [30, 31]. Given our previous finding that a proteinase which may be generally present in the stratum a recombinant truncated form of CDSN is able to bind the corneum and with a possible involvement in desquamation. Acta Derm Venereol 1991; 71: 471-4. entire CDSN [13, 14], a dominant negative interaction [3] Serre G, Mils V, Haftek M, et al. Identification of late between the mutant and wild-type proteins may account for differentiation antigens of human cornified epithelia, expressed in the loss of cohesion in the inner root sheath of the hair re-organized desmosomes and bound to cross-linked envelope. J follicles. This could affect the functionality of the inner root Invest Dermatol 1991; 97: 1061-72. sheath, such as a decrease in diffusion barrier function [4] Gallinaro H, Jonca N, Langbein L, et al. A 4.2 kb upstream region of the human corneodesmosin gene directs site-specific expression resulting in anagen to catagen transition and perturbation of in hair follicles and hyperkeratotic epidermis of transgenic mice. J a hair cycle reinitiation. However, in view of the delayed Invest Dermatol 2004; 122: 730-8. 44 The Open Dermatology Journal, 2010, Volume 4 Jonca et al.

[5] Mils V, Vincent C, Croute F, Serre G. The expression of [26] Hachem JP, Crumrine D, Fluhr J, Brown BE, Feingold KR, Elias desmosomal and corneodesmosomal antigens shows specific PM. pH directly regulates epidermal permeability barrier variations during the terminal differentiation of epidermis and hair homeostasis, and stratum corneum integrity/cohesion. J Invest follicle epithelia. J Histochem Cytochem 1992; 40: 1329-37. Dermatol 2003; 121: 345-53. [6] Simon M, Montezin M, Guerrin M, Durieux JJ, Serre G. [27] Caubet C, Jonca N, Brattsand M, et al. Degradation of Characterization and purification of human corneodesmosin, an corneodesmosome proteins by two serine proteases of the kallikrein epidermal basic glycoprotein associated with corneocyte-specific family, SCTE/KLK5/hK5 and SCCE/KLK7/hK7. J Invest modified desmosomes. J Biol Chem 1997; 272: 31770-6. Dermatol 2004; 122: 1235-44. [7] Guerrin M, Simon M, Montezin M, Haftek M, Vincent C, Serre G. [28] Bernard D, Mehul B, Thomas-Collignon A, et al. Analysis of Expression cloning of human corneodesmosin proves its identity proteins with caseinolytic activity in a human stratum corneum with the product of the S gene and allows improved extract revealed a yet unidentified cysteine protease and identified characterization of its processing during keratinocyte the so-called "stratum corneum thiol protease" as cathepsin l2. J differentiation. J Biol Chem 1998; 273: 22640-7. Invest Dermatol 2003; 120: 592-600. [8] Steinert PM, Mack JW, Korge BP, Gan SQ, Haynes SR, Steven [29] Igarashi S, Takizawa T, Takizawa T, et al. Cathepsin D, but not AC. Glycine loops in proteins: their occurrence in certain cathepsin E, degrades desmosomes during epidermal intermediate filament chains, loricrins and single-stranded RNA desquamation. Br J Dermatol 2004; 151: 355-61. binding proteins. Int J Biol Macromol 1991; 13: 130-9. [30] Matsumoto M, Zhou Y, Matsuo S, et al. Targeted deletion of the [9] Maestrini E, Monaco AP, McGrath JA, et al. A molecular defect in murine corneodesmosin gene delineates its essential role in skin loricrin, the major component of the cornified cell envelope, and hair physiology. Proc Natl Acad Sci U S A 2008; 105: 6720-4. underlies Vohwinkel's syndrome. Nat Genet 1996; 13: 70-7. [31] Leclerc EA, Huchenq A, Mattiuzzo NR, et al. Corneodesmosin [10] Sprecher E, Ishida-Yamamoto A, Becker OM, et al. Evidence for gene ablation induces lethal skin-barrier disruption and hair-follicle novel functions of the keratin tail emerging from a mutation degeneration related to desmosome dysfunction. J Cell Sci 2009; causing ichthyosis hystrix. J Invest Dermatol 2001; 116: 511-9. 122: 2699-709. [11] Terrinoni A, Puddu P, Didona B, et al. A mutation in the V1 [32] Elsayed-Ali H, Barton S, Marks R. Stereological studies of domain of K16 is responsible for unilateral palmoplantar verrucous desmosomes in ichthyosis vulgaris. Br J Dermatol 1992; 126: 24-8. nevus. J Invest Dermatol 2000; 114: 1136-40. [33] Ghadially R, Williams ML, Hou SY, Elias PM. Membrane [12] Whittock NV, Smith FJ, Wan H, et al. Frameshift mutation in the structural abnormalities in the stratum corneum of the autosomal V2 domain of human keratin 1 results in striate palmoplantar recessive ichthyoses. J Invest Dermatol 1992; 99: 755-63. keratoderma. J Invest Dermatol 2002; 118: 838-44. [34] Allen M, Ishida-Yamamoto A, McGrath J, et al. Corneodesmosin [13] Caubet C, Jonca N, Lopez F, Esteve JP, Simon M, Serre G. Homo- expression in psoriasis vulgaris differs from normal skin and other oligomerization of human corneodesmosin is mediated by its N- inflammatory skin disorders. Lab Invest 2001; 81: 969-76. terminal glycine loop domain. J Invest Dermatol 2004; 122: 747- [35] Guerrin M, Vincent C, Simon M, Ahnini TR, Fort M, Serre G. 54. Identification of six novel polymorphisms in the human [14] Jonca N, Guerrin M, Hadjiolova K, et al. Corneodesmosin, a corneodesmosin gene. Tissue Antigens 2001; 57: 32-8. component of epidermal corneocyte desmosomes, displays [36] Capon F, Novelli G, Semprini S, et al. Searching for psoriasis homophilic adhesive properties. J Biol Chem 2002; 277: 5024-9. susceptibility genes in Italy: genome scan and evidence for a new [15] Haftek M, Simon M, Kanitakis J, et al. Expression of locus on chromosome 1. J Invest Dermatol 1999; 112: 32-5. corneodesmosin in the granular layer and stratum corneum of [37] Nair RP, Stuart PE, Nistor I, et al. Sequence and haplotype analysis normal and diseased epidermis. Br J Dermatol 1997; 137: 864-73. supports HLA-C as the psoriasis susceptibility 1 gene. Am J Hum [16] Simon M, Bernard D, Minondo AM, et al. Persistence of both Genet 2006; 78: 827-51. peripheral and non-peripheral corneodesmosomes in the upper [38] Capon F, Toal IK, Evans JC, et al. Haplotype analysis of distantly stratum corneum of winter xerosis skin versus only peripheral in related populations implicates corneodesmosin in psoriasis normal skin. J Invest Dermatol 2001; 116: 23-30. susceptibility. J Med Genet 2003; 40: 447-52. [17] Lundström A, Serre G, Haftek M, Egelrud T. Evidence for a role of [39] Helms C, Saccone NL, Cao L, et al. Localization of PSORS1 to a corneodesmosin, a protein which may serve to modify desmosomes haplotype block harboring HLA-C and distinct from during cornification, in stratum corneum cell cohesion and corneodesmosin and HCR. Hum Genet 2005; 118: 466-76. desquamation. Arch Dermatol Res 1994; 286: 369-75. [40] Orrù S, Giuressi E, Carcassi C, Casula M, Contu L. Mapping of the [18] Simon M, Jonca N, Guerrin M, et al. Refined characterization of major psoriasis-susceptibility locus (PSORS1) in a 70-Kb interval corneodesmosin proteolysis during terminal differentiation of around the corneodesmosin gene (CDSN). Am J Hum Genet 2005; human epidermis and its relationship to desquamation. J Biol Chem 76: 164-71. 2001; 276: 20292-9. [41] Veal CD, Capon F, Allen MH, et al. Family-based analysis using a [19] Rawlings A. Trends in stratum corneum research and the dense single-nucleotide polymorphism-based map defines genetic management of dry skin conditions. Int J Cosmet Sci 2003; 25: 63- variation at PSORS1, the major psoriasis-susceptibility locus. Am J 95. Hum Genet 2002; 71: 554-64. [20] Jonca N, Caubet C, Guerrin M, Simon M, Serre G. [42] Simon M, Tazi-Ahnini R, Jonca N, Caubet C, Cork MJ, Serre G. Corneodesmosomal proteins. In: Rawlings AV, Leyden JJ, Eds. Alterations in the desquamation-related proteolytic cleavage of Skin moisturization, 2nd ed. New-York: Informa Healthcare USA, corneodesmosin and other corneodesmosomal proteins in psoriatic Inc; 2009; pp. 99-122. lesional epidermis. Br J Dermatol 2008; 159: 77-85. [21] Ovaere P, Lippens S, Vandenabeele P, Declercq W. The emerging [43] Capon F, Allen MH, Ameen M, et al. A synonymous SNP of the roles of serine protease cascades in the epidermis. Trends Biochem corneodesmosin gene leads to increased mRNA stability and Sci 2009 (in press). demonstrates association with psoriasis across diverse ethnic [22] Diamandis EP, Yousef GM, Luo LY, Magklara A, Obiezu CV. The groups. Hum Mol Genet 2004; 13: 2361-8. new human kallikrein gene family: implications in carcinogenesis. [44] Levy-Nissenbaum E, Betz RC, Frydman M, et al. Hypotrichosis Trends Endocrinol Metab 2000; 11: 54-60. simplex of the scalp is associated with nonsense mutations in [23] Brattsand M, Egelrud T. Purification, molecular cloning, and CDSN encoding corneodesmosin. Nat Genet 2003; 34: 151-3. expression of a human stratum corneum trypsin-like serine protease [45] Lai-Cheong JE, Arita K, McGrath JA. Genetic diseases of with possible function in desquamation. J Biol Chem 1999; 274: junctions. J Invest Dermatol 2007; 127: 2713-25. 30033-40. [46] Stefani M. Protein misfolding and aggregation: new examples in [24] Hansson L, Stromqvist M, Backman A, Wallbrandt P, Carlstein A, medicine and biology of the dark side of the protein world. Egelrud T. Cloning, expression, and characterization of stratum Biochim Biophys Acta 2004; 1739: 5-25. corneum chymotryptic enzyme. A skin-specific human serine [47] Apaydin R, Gurbuz Y, Bayramgurler D, Muezzinoglu B, Bilen N. proteinase. J Biol Chem 1994; 269: 19420-6. Cytokeratin expression in lichen amyloidosus and macular [25] Ekholm IE, Brattsand M, Egelrud T. Stratum corneum tryptic amyloidosis. J Eur Acad Dermatol Venereol 2004; 18: 305-9. enzyme in normal epidermis: a missing link in the desquamation [48] Chang YT, Liu HN, Wang WJ, Lee DD, Tsai SF. A study of process? J Invest Dermatol 2000; 114: 56-63. cytokeratin profiles in localized cutaneous amyloids. Arch Dermatol Res 2004; 296: 83-8. Corneodesmosin in Normal and Diseased Skin The Open Dermatology Journal, 2010, Volume 4 45

[49] Kobayashi H, Hashimoto K. Amyloidogenesis in organ-limited [52] Masu S, Hosokawa M, Seiji M. Amyloid in localized cutaneous cutaneous amyloidosis: an antigenic identity between epidermal amyloidosis: immunofluorescence studies with anti-keratin keratin and skin amyloid. J Invest Dermatol 1983; 80: 66-72. antiserum especially concerning the difference between systemic [50] Kumakiri M, Hashimoto K. Histogenesis of primary localized and localized cutaneous amyloidosis. Acta Derm Venereol 1981; cutaneous amyloidosis: sequential change of epidermal 61: 381-4. keratinocytes to amyloid via filamentous degeneration. J Invest [53] Baglioni S, Casamenti F, Bucciantini M, et al. Prefibrillar amyloid Dermatol 1979; 73: 150-62. aggregates could be generic toxins in higher organisms. J Neurosci [51] Maeda H, Ohta S, Saito Y, Nameki H, Ishikawa H. Epidermal 2006; 26: 8160-7. origin of the amyloid in localized cutaneous amyloidosis. Br J [54] Bucciantini M, Calloni G, Chiti F, et al. Prefibrillar amyloid Dermatol 1982; 106: 345-51. protein aggregates share common features of cytotoxicity. J Biol Chem 2004; 279: 31374-82.

Received: November 6, 2009 Revised: December 17, 2009 Accepted: December 21, 2009

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